Project Summary The path to atherosclerosis (AS) starts with vascular endothelial dysfunction (ED), which can be reversed; however, effective therapeutic strategies targeting ED are still lacking. Small RhoGTPase Rac1, an established master regulator of endothelial function, is integral to vascular homeostasis, governing vascular integrity and the nitric oxide (NO) biogenesis system, but aberrant Rac1 signaling, in particular, Rac1-triggered NADPH-oxidase (Nox)/reactive oxygen species (ROS) generation, has been strongly implicated in vascular oxidative stress and AS and may therefore be a promising target for CVD treatment. However, to be successful at determining new specific approaches to normalizing Rac1 signaling that can combat vascular oxidative stress to reverse ED and AS, we must have a deep understanding of regulatory mechanisms of Rac1 to be able to balance Rac1's critical functions with its proatherogenic consequences in endothelial cells (ECs). In this proposal, we will test whether: (1) S-glutathionylation (PrS-SG), a stable but reversible oxidative post-translational modification, on cysteine residues of Rac1 is a promising redox target, and (2) glutaredoxin-1 (Grx1), a prominent de-glutathionylation enzyme, can preserve redox status and normal signaling of Rac1. We and others reported that activity and stability of Rac1 are redox-sensitive and susceptible to PrS-SG. Our new preliminary data show that in both human aortic ECs (HAECs) and aortae from a mouse model of AS, Rac1 underwent PrS-SG and degradation in parallel with Grx1 downregulation; increasing Grx1 in HAECs preserved Rac1 redox status/expression. Increasing endothelial Grx1 in mice attenuated diet-induced ED and AS. We thus hypothesize that during AS, induction of PrS-SG on Rac1 promotes ED and AS, which can be reversed by replenishing Grx1 in endothelium. We will test three aims: (1) Characterize in vivo role of endothelial Rac1/Grx1 axis in ED and AS; (2) Elucidate the molecular mechanism of how PrS-SG on Rac1 drives ED; and (3) Test the potential of Rac1/Grx1 axis as a therapeutic target in reversing ED and AS. To define the relationship between redox status/expression/activity of Rac1/Grx1 and onset/progression of ED and AS (Aim 1), we will use EC-specific Grx1 transgenic and knockout mice, as well as pharmacological inhibition of Rac1. To test if PrS-SG on Rac1 has a causal role in ED (Aim 2), we will determine effects of expression of Rac1-SSG-mimic and -resistant mutants in generated Rac1-/- HAECs on Nox/ROS signaling, eNOS dysfunction, and hyperpermeability. To test whether replenishing endothelial Grx1 can reverse vascular dysfunction and slow AS progression (Aim 3), we will use inducible EC-Grx1 transgenic mice and ECs freshly isolated from human subjects with established AS. Success of this proposal is expected to establish a novel redox mechanism that can specifically mediate Rac1 proatherogenic signaling, and provide proof-of-concept that Grx1 can be used as a new therapeutic strategy for reversal of cardiovascular dysfunction.